CN103555844A - Fluorescent quantitative PCR (polymerase chain reaction) kit for detecting sugarcane red stripe germ - Google Patents
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Abstract
The invention relates to a fluorescent quantitative PCR (polymerase chain reaction) kit for detecting sugarcane red stripe germ. The fluorescent quantitative PCR kit comprises (1) a positive standard substance, (2) a negative control, (3) PCR reaction liquid, (4) ExTaq enzyme liquid, (5) probe solution and (6) sterile water. The kit has high specificity in detection of the sugarcane red stripe germ and has no cross reaction with other plant germs, the detection sensitivity can reach 100 copies, the kit can be directly used for quantitatively detecting sugarcane red stripe germ of samples such as sugarcane plants and detoxification seedlings, and the kit is fast and simple in detection, good in accuracy, high in sensitivity and reliable and stable in detection result.
Description
Technical field
The present invention relates to a kind of PCR test kit that detects phytopathogen, be specifically related to a kind of PCR kit for fluorescence quantitative that detects sugarcane red streak bacterium, microorganism belonging to genus detection field.
Background technology
Sugarcane red streak (Sugarcane red stripe disease) is a kind of bacterial disease generally occurring, and almost in Major Sugarcane plantation country, all has generation, and China's sugarcane red streak has fragmentary generation in several provinces that grow cane (district).Sugarcane red streak bacterium is considered to Pseudomonas rubrilineans
pseudomonas rubrilineans(Lee et al.) Stapp causes, its morphological specificity is without gemma, is shaft-like, and size is 0.7 * 1.6 μ m, has single polar flagella, has thin-walled pod film, and gramstaining reaction is negative.Willems in 1992 etc. by Pseudomonas rubrilineans rename into Acidovorax avenae subsp.avenae (
acidovorax avenaesubsp.
avenae).Zia-ul-Hussnain etc. (2011) separation from the strain of Pakistani sugarcane red streak has obtained Acidovorax avenae subsp.avenae germ, and the thalli morphology of this germ, cultivation proterties, biochemical reactions have been done to research.Its host range of Acidovorax avenae subsp.avenae germ, except sugarcane, also has the plants more gramineous such as paddy rice, corn, Herba panici repentics.Disease has two types of leaf streak and top rots.Both can occur separately, also can be concurrent.Easily morbidity under the warm condition of humidity, and often there is bacterium exudate on the scar surface of sick leaf, by gentle the spreading of rainwater, broadcasts.Pathogenic bacteria in ill withered old sugarcane leaf, sick stem still had virulence after 4 months.Therefore set up quick, accurate, the sensitive detection technique of sugarcane red streak bacterium, the control of this disease is played to very crucial effect.
The method of conventional tested for pathogens has biological assay, electron microscopy, Enzyme-multiplied immune technique and Protocols in Molecular Biology etc.Consuming time and the complex steps of biological assay; Electron microscopy needs plant tissue slice, complex steps, and can only judge by morphological observation; Enzyme-multiplied immune technique needs specific antiserum(antisera), and sensitivity is not as good as molecular detection technology; The Protocols in Molecular Biology of PCR-based principle is a kind of Fast Detection Technique of highly sensitive, high specificity, in the detection of other sugarcane pathogen, is used widely.At present, PCR kit for fluorescence quantitative and the method thereof of detection sugarcane red streak bacterium Acidovorax avenae subsp.avenae have no report.
The present invention obtains sugarcane red streak bacterium in separation, in cloning and sequencing germ genome rrna rDNA sequence basis, select sugarcane red streak bacterium 16S-23S rDNA transcribed spacer sequence (ITS), be designed for primer and the probe of fluorescence quantitative PCR detection, by the optimization to reaction system, development provides quick, sensitive, disease detection kit product and technical service accurately for detection of departments such as the PCR kit for fluorescence quantitative of sugarcane red streak bacterium and the sugarcane scientific research of method ，Wei China, quarantine, quality inspections.
Summary of the invention
The object of the invention is to provide a kind of PCR kit for fluorescence quantitative that detects sugarcane red streak bacterium, and this test kit can carry out detection by quantitative to the sugarcane red streak bacterium of testing sample fast and accurately.This test kit not only can be used in all types quantitative real time PCR Instrument in the market, and can be sensitive, special sugarcane red streak bacterium is carried out to detection by quantitative.
In order to realize object of the present invention, technical scheme of the present invention is as follows:
The present invention adopts Taqman probe to set up fluorescence quantitative PCR detection sugarcane red streak bacterium test kit, by preparation germ DNA standard model and testing sample, detects simultaneously, obtains the initial germ copy number of testing sample, and germ is carried out to detection by quantitative.
The invention provides a kind of primer pair QPCR-F5, QPCR-R5 and Taqman probe QPCR-P5 that utilizes fluorescence quantifying PCR method to detect sugarcane red streak bacterium, described primer pair sequence is as follows:
QPCR-F5：5’-TCATCCTCCACCAACCAATA-3’，
QPCR-R5：5’-TACCGGACCAACAACAAAGA-3’；
The sequence of Taqman probe QPCR-P5 is as follows:
5’-FAM-CACCAAAGCGGCTTCGCAAG-TAMRA-3’。
The present invention also provides a kind of PCR kit for fluorescence quantitative that detects sugarcane red streak bacterium, it is characterized in that this test kit is comprised of following reagent:
(1) positive criteria product: adopt DNA that conventional pcr amplification contains 16S-23S rDNA transcribed spacer as standard model, 1 * 10
10copy/μ L, 50 μ L/ bottles ,-20 ℃ of freezing preservations;
(2) negative control: adopt the Sugarcane Leaves tissue infecting without sugarcane red streak bacterium, extract the negative contrast of the total DNA of blade, 100 ng/ μ L, 50 μ L/ bottles ,-20 ℃ of freezing saving backup;
(3) PCR reaction solution: each reaction comprises 5 * PCR damping fluid, 5.0 μ L, 10 mmol dNTPs 0.5 μ L, 25 mmol MgCl
22.0 μ L and concentration are the primer pair QPCR-F5 of 10 μ mol/L and each 2.0 μ L of QPCR-R5, totally 11.5 μ L, and 50 reaction systems amount to 575 μ L, and-20 ℃ save backup;
(4) Ex Taq enzyme: each reaction system comprises 5 U/ μ L Ex Taq enzyme 0.5 μ L, 50 reaction systems amount to 25 μ L, and-20 ℃ save backup;
(5) probe solution: Taqman probe QPCR-P5 adopts 5 '-FAM and 3 '-TAMRA to modify, the synthetic rear sterilized water dilution that adopts, concentration is 10 μ mol/L, each reaction system 0.4 μ L, 50 reaction systems amount to 20 μ L, and-20 ℃ save backup;
(6) sterilized water: 2 * 10 mL/ bottles.
Advantage of the present invention and beneficial effect are mainly reflected in: this test kit has the specificity of height to the detection of sugarcane red streak bacterium, with other sugarcane germ no cross reactions; The sensitivity detecting reaches 100 copies, can be between sugarcane field detects sugarcane red streak bacterium in sample and virus-elimination seedlings equal samples; Utilize test kit of the present invention to detect simple to operate quick, from sample germ nucleic acid extraction to completing detection, 2 ~ 3 hours consuming time.
Accompanying drawing explanation
Fig. 1 is the kinetic curve of sugarcane red streak bacterium DNA standard substance quantitative fluorescent PCR.In figure, X-coordinate represents fluorescent quantitative PCR cycle number, and ordinate zou represents fluorescence signal intensity, and amplification curve is respectively DNA standard substance 1 * 10
8copy/μ L, 1 * 10
7copy/μ L, 1 * 10
6copy/μ L, 1 * 10
5copy/μ L, 1 * 10
4copy/μ L, 1 * 10
3copy/μ L, 1 * 10
2with 1 * 10
1copy/μ L.
Fig. 2 is the typical curve of sugarcane red streak bacterium DNA standard substance quantitative fluorescent PCR.X-coordinate x represents the logarithmic value of standard substance copy number, and ordinate zou y represents pcr amplification cycle number; Y=-3.2167x+39.785; R represents relation conefficient, coefficient of determination R
2=0.9998.
Embodiment
In order further to illustrate the present invention rather than restriction the present invention, below in conjunction with embodiment, be illustrated.Experimental technique described in following embodiment, if no special instructions, is ordinary method; Described reagent and biomaterial all can obtain if no special instructions from commercial channels.
1 one kinds of PCR kit for fluorescence quantitative that detect sugarcane red streak bacterium of embodiment, comprise Taqman probe QPCR-P5 and primer pair QPCR-F5, QPCR-R5, wherein Taqman probe QPCR-P5 sequence is 5 '-CACCAAAGCGGCTTCGCAAG-3 ', and 5 ' end and the 3 ' end of probe adopt respectively fluorophor FAM and TAMRA to modify; Primer pair QPCR-F5 and QPCR-R5 sequence are 5 '-TCATCCTCCACCAACCAATA-3 ' and 5 '-TACCGGACCAACAACAAAGA-3 '; This test kit comprises following reagent:
(1) positive criteria product: take 16S-F3(5 '-ACTGCGTGAAGTCGGAATC-3 ') and 23S-R1(5 '-TCGGAATCTCGGTTGATG-3 ') be primer, utilize the amplification of conventional PCR method to obtain sugarcane red streak bacterium 16S-23S rDNA transcribed spacer sequence (ITS) fragment, after PCR product purification as sugarcane red streak bacterium DNA standard model.With after nucleic acid-protein analysis-e/or determining PCR product D NA concentration, dilution is 100 ng/ μ L, and PCR product clip size is 1070 bp, and the copy number that calculates DNA standard model according to formula is 8.52 * 10
10copy/μ L, and be diluted to standard model 1 * 10
10copy/μ L, 50 μ L/ bottles ,-20 ℃ of freezing preservations.
(2) negative control: adopt the Sugarcane Leaves tissue infecting without sugarcane red streak bacterium, extract the negative contrast of the total DNA of blade, total DNA concentration of extracting with nucleic acid-protein analysis-e/or determining, and to be diluted to concentration be that 100 ng/ μ L are as negative sample, 50 μ L/ bottles ,-20 ℃ of freezing saving backup.
(3) sterilized water: 2 * 10 mL/ bottles.
(4) PCR reaction solution: each reaction comprises 5 * PCR damping fluid, 5.0 μ L, 10 mmol dNTPs 0.5 μ L, 25 mmol MgCl
22.0 μ L and concentration are the primer pair QPCR-F5 of 10 μ mol/L and each 2.0 μ L of QPCR-R5, totally 11.5 μ L, and 50 reaction systems amount to 575 μ L, and-20 ℃ save backup;
(5) Ex Taq enzyme: each reaction system comprises 5 U/ μ L Ex Taq enzyme 0.5 μ L, 50 reaction systems amount to 25 μ L, and-20 ℃ save backup;
(6) probe solution: Taqman probe QPCR-P5 adopts 5 '-FAM and 3 '-TAMRA to modify, the synthetic rear sterilized water dilution that adopts, concentration is 10 μ mol/L, each reaction system 0.4 μ L, 50 reaction systems amount to 20 μ L, and-20 ℃ save backup.
The PCR kit for fluorescence quantitative of embodiment 2 sugarcane red streak bacterium detects application
1) standard substance DNA dilution: sugarcane red streak bacterium DNA standard substance are diluted to 1 * 10 with sterilized water by 10 times
8~ 1 * 10
1a series of DNA standard substance of copy/μ L.
2) typical curve establishing equation
Take this, to dilute rear DNA standard substance be template, and each standard model is processed and repeated 3 times.And the negative contrast of the total DNA of Sugarcane Leaves to infect without sugarcane red streak bacterium, take sterilized water as blank.By component in mentioned reagent box, quantitative fluorescent PCR reaction is totally 25 μ L, comprises PCR reaction solution 11.5 μ L, reaction enzymes mixed solution 0.5 μ L, and probe solution 0.4 μ L, standard model DNA 1.0 μ L, sterilized water is mended to 25 μ L; Then be put in ABI company 7500 quantitative PCR instrument and carry out fluorescent quantitative PCR.Amplification program is: 95 ℃ of 15 s, and 60 ℃ of 1 min, 40 circulations, carry out single-point fluoroscopic examination at 60 ℃.Obtain amplification curve as shown in Figure 1, then draw typical curve as shown in Figure 2, build typical curve equation.The typical curve equation that the present embodiment builds is Y=-3.2167x+39.785, coefficient of determination R
2be 0.9998, amplification efficiency is 104.58 %, meets the normal typical curve requirement of quantitative fluorescent PCR.Detection sensitivity can 10 copy/μ L.Fluorescence quantifying PCR method is obtained to the conventional electrophoresis detection of product, can only detect 10
4copy/μ L is at least higher 100 times than conventional PCR detection sensitivity.
3) testing sample DNA extraction: adopt CTAB method to extract several parts of Sugarcane Leaves DNA to be measured, measure the absorbance value of DNA on nucleic acid-protein analyser, calculate the DNA concentration of extracting, then the unified 100 ng/ μ L that are diluted to.
4) fluorescent quantitative PCR: the RNA of testing sample of take is template, quantitative fluorescent PCR reacts total system and amplification program with above-mentioned typical curve establishing equation.ABI company 7500 quantitative PCR instrument carry out fluorescent quantitative PCR, obtain the kinetic curve of the sugarcane red streak bacterium standard substance quantitative fluorescent PCR of testing sample.The Ct value substitution typical curve equation of testing sample is changed into initial germ molecule copy number, and a molecule copy number represents a sugarcane red streak bacterium, thus the quantity that infects sugarcane red streak bacterium in quantitative analysis sugarcane sample, and result is as shown in table 1.Detected result shows, No. 38, good fortune agriculture, good fortune agriculture 1110 these 2 field samples with typical sugarcane red streak symptom confirm to have infected sugarcane red streak bacterium, and germ content reaches 10
5copy/μ L; Do not have new platform sugar 25, these 2 field samples of Dehong 06-24 of sugarcane red streak symptom not to detect sugarcane red streak bacterium.In order further to confirm whether the germ that No. 38, good fortune agriculture, good fortune agriculture 1110 are infected is sugarcane red streak bacterium, we carry out Cloning and sequencing by quantitative fluorescent PCR product, nucleotide sequence is submitted http://blast.ncbi.nlm.nih.gov/ comparison to, result confirms it is sugarcane red streak bacterium, belong to Acidovorax avenae subsp.avenae (
acidovorax avenaesubsp.
avenae).The detection sugarcane red streak bacterium fluorescence quantifying PCR method that the present invention sets up has good repeatability, and the Ct value variation coefficient of same sample is less than 1 %.
While utilizing Taqman fluorescence probe quantitative PCR test kit of the present invention to detect, the sugarcane red streak bacterium initial copy number of sample can be obtained quickly and accurately, sample detection between sugarcane field, virus-elimination seedlings quality monitoring and sugarcane Resistance Identification can be applied to.
Table 1 testing sample Ct value and stability
<110> University Of Agriculture and Forestry In Fujian
<120> PCR kit for fluorescence quantitative that detects sugarcane red streak bacterium
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> artificial sequence
<400> 1
<210> 2
<211> 20
<212> DNA
<213> artificial sequence
<400> 2
<210> 3
<211> 24
<212> DNA
<213> artificial sequence 3
<400> 3
caccatcacg aaatcaatgc agca 24
Claims (2)
1. a PCR kit for fluorescence quantitative that detects sugarcane red streak bacterium, comprise Taqman probe QPCR-P5 and primer pair QPCR-F5, QPCR-R5, wherein Taqman probe QPCR-P5 sequence is 5 '-CACCATCACGAAATCAATGCAGCA-3 ', and 5 ' end and the 3 ' end of probe adopt respectively fluorophor FAM and TAMRA to modify; Primer pair QPCR-F5 and QPCR-R55 sequence are 5 '-CGGGAAACCCATAATACCAC-3 ' and 5 '-GTCGATTTCTGCTGGTGAGA-3 '.
2. for a PCR kit for fluorescence quantitative for sugarcane red streak bacterium, it is characterized in that this test kit is comprised of following reagent:
(1) positive criteria product: adopt DNA that conventional pcr amplification contains 16S-23S rDNA transcribed spacer as standard model, 1 * 10
10copy/μ L, 50 μ L/ bottles ,-20 ℃ of freezing preservations;
(2) negative control: adopt the Sugarcane Leaves tissue infecting without sugarcane red streak bacterium, extract the negative contrast of the total DNA of blade, 100 ng/ μ L, 50 μ L/ bottles ,-20 ℃ of freezing saving backup;
(3) PCR reaction solution: each reaction comprises 5 * PCR damping fluid, 5.0 μ L, 10 mmol dNTPs 0.5 μ L, 25 mmol MgCl
22.0 μ L, and primer pair claimed in claim 1, concentration is the QPCR-F5 of 10 μ mol/L and each 2.0 μ L of QPCR-R5, totally 11.5 μ L, 50 reaction systems amount to 575 μ L, and-20 ℃ save backup;
(4) Ex Taq enzyme: each reaction system comprises 5 U/ μ L Ex Taq enzyme 0.5 μ L, 50 reaction systems amount to 25 μ L, and-20 ℃ save backup;
(5) probe solution: Taqman probe QPCR-P5 as claimed in claim 1, adopts 5 '-FAM and 3 '-TAMRA to modify, the synthetic rear sterilized water dilution that adopts, concentration is 10 μ mol/L, each reaction system 0.4 μ L, 50 reaction systems amount to 20 μ L, and-20 ℃ save backup;
(6) sterilized water: 2 * 10 mL/ bottles.
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| CN106222291A (en) * | 2016-08-19 | 2016-12-14 | 福建农林大学 | A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium |
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| CN1712538A (en) * | 2004-06-25 | 2005-12-28 | 中国农业科学院植物保护研究所 | Rapid detection of bacterial fruit spot germ of melon seed |
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| US6146834A (en) * | 1999-09-10 | 2000-11-14 | The United States Of America As Represented By The Secretary Of Agriculture | PCR primers for detection of plant pathogenic species and subspecies of acidovorax |
| CN1712538A (en) * | 2004-06-25 | 2005-12-28 | 中国农业科学院植物保护研究所 | Rapid detection of bacterial fruit spot germ of melon seed |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106222291A (en) * | 2016-08-19 | 2016-12-14 | 福建农林大学 | A kind of Molecular Detection and the test kit identifying the Caulis Sacchari sinensis different strain of red streak bacterium |
| CN106222291B (en) * | 2016-08-19 | 2019-06-28 | 福建农林大学 | A kind of kit of Molecular Detection strain different with identification sugarcane red streak bacterium |
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| CN103555844B (en) | 2014-11-26 |
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